gms | German Medical Science

Objective: Effective ways of pharmacological neuroprotection are one of greatest goals in modern medicine. The influx of calcium is widely regarded as a final common pathway in neuronal cell death. We postulated that combined inhibition of L-type calcium channels and the acid-sensing ion channel 1a (ASIC1a) using the two clinically established drugs nimodipine and amiloride may result in neuroprotection and thus reduced cell death using a well-established in-vitro model of cerebral ischemia.

Methods: Hippocampus-slices from 4-7 days old mice pups (C57BL/6N) were prepared and cultivated in growth medium at 37°C and 5% CO2 for 14 days. Baseline images for cell death assessment were obtained using propidium iodid (PI) staining and fluorescence microscopy. Slices then underwent oxygen-glucose deprivation (with 95% N2, 5% CO2) for 60 minutes. Afterwards slices received no treatment (control group; n=101), 2 Vol.% dimethyl-sulfoxide (DMSO) (solvent control; n=45) or treatment consisting of either 100µM amiloride alone (n=43) or a combination with 10µM (n=47) or 20µM (n=46) nimodipine. After incubation for 72h cell death was assessed using the pre-described PI staining and fluorescence microscopy. We used a Kruskal-Wallis test to calculate p-values and posthoc Dunn’s Test (using GraphPad Prism Version 8.2.0) to correct for multiple comparisons, with p<0.05 regarded as statistically significant

Results: Slices receiving only DMSO as vehicle or additional amiloride did not show any statistically significant differences in levels of cell death. Slices receiving the combination of 100µM amiloride and 10µM or 20µM nimodipine respectively at 2.0 Vol.% DMSO displayed a significant increase in cell damage levels at 72 hours after OGD compared to the OGD control group (p=0.0001 for both).

Conclusion: No effective neuroprotection could be observed using a combination of nimodipine and amiloride. Interestingly, the combination of the two drugs statistically impaired cell outcome after OGD. A possible explanation might be unspecific effects of amiloride combined with increased extracellular calcium levels through L-type calcium blockage. Our results suggest that the simultaneous administration of nimodipine and amiloride (e.g. during the treatment of delayed cerebral ischemia) should be critically evaluated.

Figure 1 [Fig. 1]